qPCR
Reverse transcription of 1 μg of RNA into cDNA was performed using the
QuantiTect Reverse Transcription Kit (Qiagen). The qPCR was performed
using QuantiFast SYBR Green PCR kit (Qiagen) in the presence of 0.5 μM
primers in a CFX96 thermocycler (Biorad, Hercules, CA, USA) following
the manufacturer’s instructions. PCR conditions were as follows:
incubation at 95 °C for 5 min, 45 cycles 10 s 95°C and 30 s 60°C.
Additionally melting curve analysis was performed at the end of each run
to ensure specificity of the products. The same master mix without cDNA
was used as negative control. The following primers were used: WHY1
(AK365452.1) Fwd 5´-GATGGGAATGGTCGCTTTTT -3´, Rev
5´-CCATGATGTGCGGTATGATG -3´), ACTIN 11 (AY145451) Fwd
5’- CGACAATGGAACCGGAATG-3’, Rev 5’-CCCTTGGCGCATCATCTC-3’) and Elongation
factor 1- a (Z50789) Fwd 5´-TTGGTGGCATTGGAACTGTG -3´Rev
5’-CAAACCCACGCTTGAGATCC-3´.